In recent years, national and international attention has become focused on the problem of the release into the environment of chemical reagents that are potential health hazards. The problem is complicated by the socio-economic aspects of production and use of these chemicals and by our limited knowledge of the long term effects of such reagents, with regard to their carcinogenicity and mutagenicity. The public obviously cannot afford to wait five to twenty five years after the introduction of a new reagent to see if it is a health hazard. Methods for evaluation or screening of potentially hazardous materials are therefore needed that provide an indication of the degree of hazard in relatively short time periods.
There have been a number of approaches to evaluation and/or screening of chemicals to evaluate their hazard potential. Since the problem is one of the effect of chemicals on biological entities, the approaches have centered on bioassay procedures. The range of biota chosen for controlled exposure to potentially hazardous chemicals has been enormous. On one end of the spectrum are single cells such as bacteria yeast and mammalian cells. At the other end are multifunctional organisms, such as plants and animals. In general, the tendency has been towards screening with less biologically complex entities, followed by more definitive confirmation testing with complex biota of those chemicals found hazardous in the screening effort.
Among the generally accepted screening procedures for carcinogens and mutagens is the so-called Ames test developed by Dr. Bruce N. Ames and his associates. The Ames test is based upon the assumption that carcinogens will cause the genetic reversion of certain mutant strains of bacteria such as Salmonella typhimurium. In other words, the mutant strains revert to their normal or "wild" form in the presence of carcinogens and mutagens. Since the majority of mutagens or their metabolic products are also carcinogens, the Ames test is also used to screen this class of chemicals. Extensive testing with a wide variety of potential carcinogens has indicated the general validity of the basic assumption that carcinogens cause bacterial reversion.
Basically, the mutant Salmonella typhimurium strains are selected because they lack the ability to produce histidine, an essential amino acid. These mutants are unable to multiply unless this essential nutrient is present in their growth media. In the presence of carcinogens and mutagens, the mutated strains revert to their "wild" form. Since the "wild" forms can manufacture histidine from other materials, it need not be present in the growth media in order for the reverted strains to multiply. Thus, by culturing mutant strains of Salmonella typhimurium in a media that does not contain histidine or contains a minimal amount of histidine, but does contain a suspected chemical, one can evaluate the carcinogenicity or mutagenicity by determining the growth charactristics. It is not, however, a go, no-go situation, since there is a certain portion of the mutant bacteria that reverts spontaneously, without any experimental stimulus. This "natural" reversion rate phenomena represents a "noise level" that limits the overall sensitivity of the test. It is not a critical factor with properly chosen bacterial strains, i.e. those with low spontaneous reversion rates.
As with most bacterial tests, plate counting techniques are used to determine the number of revertant bacteria. For the Ames test, plate counting requires a 48 hour growth period, plus time to count both the test plates and control plates used to monitor spontaneous reversion. The Ames test is discussed more fully in an article by Ames et al appearing in Mutation Research, Volume 31 (1975) pages 347-364.
It is an object of the present invention to provide a rapid screening of potential carcinogens or mutagens. It is a further object to reduce the effort required for such screening. It is still a further object of the invention to provide suitable apparatus for carrying out this method.